The progression of a normal cell to a fully malignant tumor is a multistep process involving interactions between external mutagenic or growth promoting stimuli and the activation of one or more members of a diverse set of cellular genes called oncogenes. Oncogene-mediated multistep transformation has been demonstrated in primary cell cultures but is not a general property of established cell lines. The C3H1OT1/2 cell line is an exception and behaves similarly to primary cells when transfected with the human H-ras oncogene and the MC29 viral myc oncogene. Ras/myc cooperation in C3H1OT1/2 fibroblasts is marked by an increase in focus formation and a highly transformed cell morphology that reflects an increased potential for anchorage independent growth in soft agarose. To assess the contribution of each oncogene to the ras/myc transformed phenotype, C3H1OT1/2 cell lines expressing ras, myc or both oncogenes will be examined for levels of oncogene expression and assayed for tumorigenicity in nude mice. The increase in focus formation following co-transfection of C3H1OT1/2 cells with ras and myc, coupled with site-directed mutagenesis of the MC29 gag- myc gene, will be used to identify the myc sequences important to ras/myc cooperative transformation and also to identify the sequences specifying nuclear localization of the gag-myc protein. The temporal relationship between ras expression and myc expression in cooperative transformation will be explored in time-course studies using an inducible c-myc C3H1OT1/2 cell line. In an effort to dissect further the molecular intermediates in the pathway of ras/myc cooperation in C3H1OT1/2 cells, we will prepare cDNA libraries from C3H1OT1/2 and C3H1OT1/2 myc cells and differentially screen the libraries for messages up-regulated or down-regulated in response to myc over-expression. The cDNAs identified will be examined for the appropriate pattern of myc-related expression in various cell lines. Full length cDNAs will be subcloned into expression vectors and transfected into C3H1OT1/2 cells for functional assays. The experiments outlined in this proposal are designed to increase our knowledge of myc function in mammalian cells and to identify additional genes whose products are important to multistep transformation in culture and the progression of tumor formation in vivo.